Towards mastering CRISPR-induced gene knock-in in plants: Survey of key features and focus on the model Physcomitrella patens

Abstract

a b s t r a c tBeyond its predominant role in human and animal therapy, the CRISPR-Cas9 system has also become an essential tool for plant research and plant breeding. Agronomic applications rely on the mastery of gene inactivation and gene modification. However, if the knock-out of genes by non-homologous end-joining (NHEJ)-mediated repair of the targeted double-strand breaks (DSBs) induced by the CRISPR-Cas9 system is rather well mastered, the knock-in of genes by homology-driven repair or end-joining remains difficult to perform efficiently in higher plants. In this review, we describe the different approaches that can be tested to improve the efficiency of CRISPR-induced gene modification in plants, which include the use of optimal transformation and regeneration protocols, the design of appropriate guide RNAs and donor templates and the choice of nucleases and means of delivery. We also present what can be done to orient DNA repair pathways in the target cells, and we show how the moss Physcomitrella patens can be used as a model plant to better understand what DNA repair mechanisms are involved, and how this knowledge could eventually be used to define more performant strategies of CRISPR-induced gene knock-in.

A theophylline-responsive riboswitch regulates expression of nuclear-encoded genes in Arabidopsis

“…Currently, targeted genome editing in higher plants is limited to gene disruption whereas efficient and reliable methods for precise insertion of DNA constructs into the nuclear genome are still missing (Collonnier et al, 2017). Therefore, there is no straightforward possibility to regulate endogenous plant genes by introduction of artificial riboswitches.…”

SPIRAL2 Stabilises Endoplasmic Microtubule Minus Ends in the Moss <i>Physcomitrella patens</i>

“…Homologous recombination was confirmed by PCR as described in Fig. 1B, C. To generate CRISPR-mediated frameshift mutations 4 (Collonnier et al, 2017), 20 bp guide RNAs (gRNAs) targeting the start of SPR2a, SPR2b, SPR2c, SPR2d genes were designed using CRISPOR (http://crispor.tefor.net/). Forward and reverse oligonucleotides bearing the 20 bp gRNA template sequences with sticky ends were ligated into the BsaI site in pCasGuide/pUC18.…”

“…Streptococcus pyogenes Cas9 expression plasmid (Collonnier et al, 2017). pSY034 pCasGuide/pUC18 (Collonnier et al, 2017) was modified to replace the BsaI site with MfeI and ClaI sites. The resulting plasmid was further modified to include a nourseothricin resistance expression cassette.…”

Transient cotransformation of CRISPR/Cas9 and oligonucleotide templates enables efficient editing of target loci in Physcomitrella patens

“…However, it is unclear whether hypomorphic mutations can be introduced when a template is supplied. Moreover, template-free editing can induce specific deletions at a high frequency, which may complicate the identification of out-of-frame mutations.Oligodeoxynucleotides (ODNs) and CRISPR/Cas9 have been used in combination to achieve mutation knock-in in flowering plants (Collonnier et al, 2017b). We asked whether the same strategy can be applied to P. patens.…”

“…Oligodeoxynucleotides (ODNs) and CRISPR/Cas9 have been used in combination to achieve mutation knock-in in flowering plants (Collonnier et al, 2017b). We asked whether the same strategy can be applied to P. patens.…”